Sleep Loss Can Cause Death Through Accumulation of Reactive Oxygen Species in The Gut

Article
Sleep Loss Can Cause Death through Accumulation

of Reactive Oxygen Species in the Gut
Graphical Abstract Authors
Alexandra Vaccaro, Yosef Kaplan Dor,
Keishi Nambara, Elizabeth A. Pollina,
Cindy Lin, Michael E. Greenberg,
Dragana Rogulja
Correspondence
dragana_rogulja@hms.harvard.edu
In Brief
Sleep deprivation is associated with
lethality through accumulation of reactive
oxygen species in the gut.
Highlights
d Sleep deprivation leads to ROS accumulation in the fly and
mouse gut
d Gut-accumulated ROS trigger oxidative stress in this organ
d Preventing ROS accumulation in the gut allows survival

without sleep in flies
Vaccaro et al., 2020, Cell 181, 1–22

June 11, 2020 ª 2020 Elsevier Inc.
https://doi.org/10.1016/j.cell.2020.04.049 ll
Please cite this article in press as: Vaccaro et al., Sleep Loss Can Cause Death through Accumulation of Reactive Oxygen Species in the Gut,
Cell (2020), https://doi.org/10.1016/j.cell.2020.04.049
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Article
Sleep Loss Can Cause Death through Accumulation
of Reactive Oxygen Species in the Gut
Alexandra Vaccaro,1,2 Yosef Kaplan Dor,1,2 Keishi Nambara,1 Elizabeth A. Pollina,1 Cindy Lin,1 Michael E. Greenberg,1
and Dragana Rogulja1,3,*
1Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA
2These authors contributed equally
3Lead Contact
*Correspondence: dragana_rogulja@hms.harvard.edu
https://doi.org/10.1016/j.cell.2020.04.049
SUMMARY
The view that sleep is essential for survival is supported by the ubiquity of this behavior, the apparent exis-
tence of sleep-like states in the earliest animals, and the fact that severe sleep loss can be lethal. The cause of
this lethality is unknown. Here we show, using flies and mice, that sleep deprivation leads to accumulation of
reactive oxygen species (ROS) and consequent oxidative stress, specifically in the gut. ROS are not just cor-
relates of sleep deprivation but drivers of death: their neutralization prevents oxidative stress and allows flies
to have a normal lifespan with little to no sleep. The rescue can be achieved with oral antioxidant compounds
or with gut-targeted transgenic expression of antioxidant enzymes. We conclude that death upon severe
sleep restriction can be caused by oxidative stress, that the gut is central in this process, and that survival
without sleep is possible when ROS accumulation is prevented.
INTRODUCTION bolic, and circulatory systems (Ali et al., 2013; Irwin, 2019; Kha-
nijow et al., 2015; Koren and Taveras, 2018; McAlpine et al.,
Sleep is an all-encompassing state that renders animals immo- 2019; Mullington et al., 2010; Tobaldini et al., 2019). It is unclear
bile and hypo-responsive to stimulation (Campbell and Tobler, whether these are secondary consequences of altered nervous
1984). A single answer is unlikely to explain the function of sleep system function or direct and independent effects of sleep depri-
since processes like cognition, immunity, and metabolism are all vation. It is also unclear whether any of these impairments
sleep dependent (Krueger et al., 2016; Zielinski et al., 2016). contribute to premature death of sleep-deprived animals. Given
Countless clinical and experimental studies link insufficient sleep the diverse system failures, it is important to know whether a sin-
with serious health problems (Chattu et al., 2018; Medic et al., gle intervention could prevent lethality in sleep-deprived animals
2017), and sleep restriction can lead to premature death in model or whether the longevity benefits of rescuing one problem would
organisms, including dogs, rats, cockroaches, and flies (Benti- be masked by a host of other consequences incompatible with
voglio and Grassi-Zucconi, 1997; Rechtschaffen et al., 1983; life. Prevention of death by a single means would argue that
Shaw et al., 2002; Stephenson et al., 2007). We wanted to find the gradual collapse of nearly all major bodily functions derives
out specifically what makes sleep required for survival in the from a common origin.
most basic sense. One proposed function of sleep is prevention of oxidative
Sleep is generated by neurons, so it has been assumed that stress in the brain (Reimund, 1994). Multiple studies have re-
death observed with sleep deprivation results from impaired ported altered antioxidant response in the brain during sleep
brain function. This idea is supported by the significant cognitive loss (Alzoubi et al., 2012; D’Almeida et al., 1998; Hill et al.,
decline noticeable after sleep loss (Donlea, 2019; Killgore, 2010; 2018; Kanazawa et al., 2016; Ramanathan et al., 2002; Silva et
Krause et al., 2017). Two leading proposals for the brain-related al., 2004; Singh and Kumar, 2008; Süer et al., 2011; Villafuerte
restorative functions of sleep are downscaling of synapses et al., 2015), and it was recently found that sleep loss alters the
formed during wakefulness (Bushey et al., 2011; de Vivo et al., redox state of several sleep-regulating neurons in the fly brain,
2017; Diering et al., 2017; Gilestro et al., 2009; Tononi and Cirelli, influencing their activity (Kempf et al., 2019). Because the brain
2014) and clearance of harmful substances from interstitial brain does not appear to be significantly damaged by sleep depriva-
areas (Holth et al., 2019; Xie et al., 2013). Because it is difficult to tion (Cirelli, 2006; Cirelli et al., 1999; de Souza et al., 2012; Eiland
induce these processes during wakefulness or inhibit them dur- et al., 2002; Hipólide et al., 2002), some labs have searched for
ing sleep, their contribution to the lethality associated with sleep signs of oxidative stress elsewhere. In rodents, the endogenous
deprivation is unknown. In addition to impairing cognition, sleep antioxidant defense is weakened in the liver (Everson et al., 2005;
loss leads to dysfunction of the gastrointestinal, immune, meta- Pandey and Kar, 2018), and visceral organs show evidence of
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oxidation after long-term sleep restriction (Everson et al., 2014; RESULTS

Pandey and Kar, 2018; Villafuerte et al., 2015). As there are no
known localized origins of oxidative molecules during sleep Severe Sleep Loss Can Cause Premature Death
deprivation, it is not known whether this oxidation is a cause or To study how severe sleep loss impacts survival, we looked for
consequence of other damage and whether it is the reason methods that can continually suppress most sleep. It is difficult
why sleep-deprived animals die. to keep animals awake for a long time because a powerful ho-
In our search for factors that directly link sleep loss and death, meostatic mechanism drives recovery of lost sleep; this
we took an agnostic approach in terms of anatomy, examining compensatory mechanism is not well understood but is evident
multiple tissues in parallel. Our first model was the fly since flies in rebound sleep seen after a night of sleep deprivation (i.e., an-
and mammals share core attributes of sleep (Hendricks et al., imals sleep instead of being active) (Allada et al., 2017; Hen-
2000; Nitz et al., 2002; Shaw et al., 2000) and since flies require dricks et al., 2000; Huber et al., 2004; Shaw et al., 2000). As
sleep for a normal lifespan (Bushey et al., 2010; Cirelli et al., can be intuited (Bringmann, 2019), to reveal the full consequence
2005a; Koh et al., 2008; Pitman et al., 2006; Rogulja and Young, of sleep loss, it is likely necessary to bypass the mechanisms that
2012; Shaw et al., 2002; Stavropoulos and Young, 2011). After drive rebound sleep. Crucially, lack of such drive should not elim-
determining how long it takes for sleep deprivation to cause inate the physiological need for sleep, the same way that a lack
death, we examined various markers of cell damage in the of appetite would not eliminate the need for nutrients.
days leading up to that point. We identified reactive oxygen spe- A common sleep deprivation method in flies relies on thermo-
cies (ROS)—unstable, short-lived, and highly reactive mole- genetic stimulation of various neurons whose activity sup-
cules—as drivers of cellular damage and lethality during sleep presses sleep (Dubowy et al., 2016; Kayser et al., 2014, 2015;
deprivation. Endogenously generated ROS have important Liu et al., 2012, 2016; Nall et al., 2016; Seidner et al., 2015; Si-
signaling roles (Holmström and Finkel, 2014; Li et al., 2018; taraman et al., 2015; Vienne et al., 2016). We tested most Gal4
Lim et al., 2014; Mittler, 2017; Oswald et al., 2018; Owusu-Ansah drivers known to target such neurons, using them to express
and Banerjee, 2009; Schieber and Chandel, 2014), but when the heat-activated cation channel TrpA1 (Hamada et al., 2008).
their levels exceed the cellular antioxidant capacity, a chain of re- At 21 C, TrpA1 is closed, so this temperature was used to raise
actions is triggered that results in widespread oxidation (D’Au- animals and monitor baseline sleep. Increasing the ambient tem-
tréaux and Toledano, 2007; Schieber and Chandel, 2014). The perature to 29 C causes TrpA1 to open and neurons to be stim-
free radical forms of ROS are particularly reactive because their ulated as a consequence. Sleep was reduced with all Gal4s, but
valence electrons are unpaired (Dhawan, 2014). To gain stability, in most cases, animals could not be kept awake for more than
they interact with macromolecules (DNA, proteins, lipids), strip- several days (data not shown). Fortunately, some Gal4s have
ping them of electrons and consequently destabilizing them been shown to target neurons whose activation causes severe
(Evans et al., 2004; Gutteridge and Halliwell, 1990; Halliwell, sleep loss without triggering a compensatory increase in sleep
2006; Stadtman and Levine, 2003). drive; when deprivation is stopped, animals resume normal
Three independent methods of sleep deprivation in the fly led sleep-wake cycles without seeking extra sleep (Seidner et al.,
to accumulation of ROS in the gut, triggering oxidative stress in 2015). Consistent with this characterization, such Gal4s were
this organ. When deprivation was stopped, ROS and oxidative effective at suppressing sleep through life (Figure 1). We used
stress markers gradually cleared. Sleep deprivation in the two Gal4s in parallel (Figures 1A–1D) to increase our confidence
mouse produced a similar outcome, with ROS accumulating that whatever phenomena we observe are generalizable. The
specifically in the small and large intestines and triggering conclusions we reached with this approach were later supported
oxidative stress. We show in flies that accumulated ROS are by other methods of deprivation, including those that trigger
causally linked to decreased survival, as clearing them from sleep rebound.
the gut (with oral antioxidant compounds or gut-targeted When TrpA1 was expressed in neurons labeled by 11H05- or
expression of antioxidant enzymes) can allow a normal lifespan 60D04-Gal4 (11H05>TrpA1, 60D04>TrpA1), flies lost 90% of
with little to no sleep. sleep (Figures 1A–1D). In each case, sleep-deprived animals
Figure 1. Sleep Deprivation Shortens Lifespan

(A) Expression of 11H05-Gal4 in the nervous system, reported by mCD8::GFP. Top: brain. Bottom: ventral nerve cord. Brp, a presynaptic protein, marks the
neuropil.
(B) Sleep across 3 days. Day 1, 21 C: sleep amount is the same between the parental controls (11H05-Gal4, UAS-TrpA1) and flies expressing TrpA1
(11H05>TrpA1). Male flies sleep in the middle of the day and at night. When the temperature is raised to 29 C on day 2 (orange), 11H05>TrpA1 flies become nearly
sleepless. Mean and SEM (shading). Right: daily sleep in control and experimental flies. Mean and SEM.
(C and D) The same as (A) and (B) but for 60D04-Gal4. Scale bars, 200 mm.
(E) Survival of sleep-deprived (green, blue) and non-deprived flies at 29 C. Left: population. Right: individual survival. Mean and SEM.
(F) Correlation between sleep and lifespan in individuals.
(G) Left: survival of flies allowed to sleep after being deprived for 10 days. Error bars are omitted for clarity. Center and right: sleep during recovery. Mean
and SEM.
(H) Flies were sleep-deprived for 10 days (SD1) and allowed to sleep for 5, 10, or 15 days before being deprived again (SD2). Day 0, onset of SD2. Error bars are
omitted for clarity.
All survival experiments were performed at least twice. For sample sizes and statistical analyses, see Table S1. See also Figure S2.
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died earlier than their non-deprived parental controls (Figure 1E). peroxide (Cathcart et al., 1983), also reported an increase in
High mortality became apparent around day 10 of deprivation ROS levels specifically in the gut (Figure S1A).
(Figures 1E and 1F). Greater sleep loss triggered earlier death ROS accumulation was gradual, peaking on day 10 of sleep
in individual flies (Figure 1F). If deprivation was stopped on day deprivation (Figures 2E, top row, and 2F and data not shown).
10, the surviving animals established normal sleep-wake cycles When deprivation was stopped, ROS levels gradually decreased
and lived as long as the controls (Figure 1G). The lifespan of flies (Figures 2E, bottom row, and 2F), coming close to baseline after
extends at lower temperatures (21 C versus 29 C); regardless, 15 days (Figure 2F), which is also how long it took for survival
there was no difference in survival between the experimental ge- curves to reset after sleep deprivation was stopped (Figure 1H).
notypes and the controls (Figure 1G, left), suggesting that at least ROS levels remained slightly elevated relative to pre-deprivation
some major negative consequences of sleep deprivation are levels even after 20 days of recovery (data not shown).
reversible. The two Gal4 lines used for sleep deprivation have almost no
To find out how long the impact of sleep deprivation lingers in expression in the gut, except for several cells that can be de-
the body, we deprived flies of sleep for 10 days, allowed different tected with a bright fluorescent nuclear marker (Barolo et al.,
groups of animals to sleep ad libitum for different time durations, 2004; Figure S1B). Still, we ensured that our observations did
and then restricted their sleep again. Being allowed to sleep for 5 not stem from TrpA1-dependent activation of gut cells. Expres-
or 10 days was insufficient to erase the impact of the first bout of sion and activation of TrpA1 in different cell populations in the gut
deprivation (Figure 1H, first two graphs). If the period of unre- (enterocytes, using myo1A-gal4 [Buchon et al., 2013; Jiang et al.,
stricted sleep was extended to 15 days, flies seemed fully recov- 2009], or enteroblasts and intestinal stem cells, using esg-Gal4
ered (Figure 1H, last graph), as it took 20 days before they all died [Micchelli and Perrimon, 2006]) did not affect sleep, longevity,
(comparable with Figure 1E). These results suggest a gradual or ROS levels (Figures S1C–S1H). We conclude that sleep depri-
accumulation of damage during sleep loss that is also gradually vation is the most likely cause of ROS increase in the gut. To
repaired during recovery sleep. strengthen this conclusion and ensure that ROS accumulation
is not limited to methods that do not trigger sleep rebound, we
Thermogenetic Sleep Deprivation Causes Accumulation used additional approaches to prevent sleep.
of ROS in the Gut
Because mortality increased around day 10 of sleep deprivation Other Sleep Deprivation Methods Also Lead to ROS
(Figures 1E, 1F, and 2A), we examined various markers of cell Accumulation in the Gut
damage in the preceding days, throughout the body. Most tis- Several modes of mechanical agitation have been used for
sues appeared indistinguishable between sleep-deprived and acute, short-term sleep deprivation in Drosophila (Hendricks
non-deprived animals, except for one major difference: the et al., 2000; Huber et al., 2004; Kayser et al., 2014; Li et al.,
guts of deprived animals had increased levels of ROS (Figures 2009; Seugnet et al., 2008; Shaw et al., 2000, 2002). Exposing
2B and 2C). High ROS levels were mostly observed in the flies to vibrations did cause sleep reduction, but with high vari-
midgut, which spans much of the gut length but has clear bound- ability across individuals (Figures 3A and 3D) and across days
aries (Figure 2D; Miguel-Aliaga et al., 2018). ROS are inherently (Figure 3B). The variability was presumably due to habituation
unstable because of high reactivity and are consequently difficult but also the fact that the compensatory sleep drive increases
to detect. This problem can be circumvented with probes that in response to mechanical deprivation (Hendricks et al., 2000;
form stable fluorescent products when oxidized by ROS (Chen Huber et al., 2004; Shaw et al., 2000). Consistent with this inter-
et al., 2011; Hardy et al., 2018). Dihydroethidium (DHE) is consid- pretation, rebound sleep was observed immediately after the
ered the most sensitive and specific among such probes and is agitation was stopped (Figure 3C). The extreme variability of
used to detect superoxide radicals in living (non-fixed) tissues. sleep loss, and the fact that wings and legs were often damaged
DHE emits blue fluorescence unless oxidized, in which case it in- by shaking, made it difficult to correlate the observed shortening
tercalates into DNA and fluoresces red (Kalyanaraman et al., of lifespan (data not shown) with sleep loss. Despite this limita-
2017). Superoxide is a precursor of other ROS (Babior, 1997; Fri- tion, we could ask whether sleep loss induced by mechanical
dovich, 1997), so DHE fluorescence should probably be taken as deprivation leads to ROS accumulation. Most flies exhibited sig-
a general reporter of reactive species and free radicals. Staining nificant sleep loss on the first day, but their sleep increased sub-
was performed on whole tissues because their small size allows sequently (e.g., Figure 3A, inconsistent sleep loss). A minority of
DHE penetration (Vaccaro et al., 2017). A different fluorescent flies consistently lost most of their sleep for up to a week (e.g.,
probe, thought to be predominantly oxidized by hydrogen Figure 3A, consistent sleep loss). Because they experienced
Figure 2. ROS Accumulate in the Fly Gut upon Prolonged Thermogenetic Sleep Deprivation
(A) Survival of sleep-deprived flies (green, blue) and non-deprived parental controls, initial 12 days. Performed 3 times. Mean and SEM.
(B) Oxidized DHE (DHE ox) reports high levels of reactive oxygen species (ROS) in the gut of sleep-deprived flies (green, blue) on day 10.
(C) Quantification. Mean and SEM.
(D) Closeup of the gut on day 10 of sleep deprivation. The same image as in (B) (11H05>TrpA1).
(E) Top: ROS in the gut on days 1, 7, and 10 of sleep deprivation (29 C). Bottom: ROS in the gut 5, 10, or 15 days after deprivation was stopped (21 C).
Representative images. Scale bars, 200 mm.
(F) Quantification. Mean and SEM.
For sample sizes and statistical analyses, see Table S1. See also Figures S1 and S2.
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identical mechanical agitation but different sleep amounts, ani- The one verified example of severely sleep-deprived flies with
mals could be directly compared to reveal the effect of sleep normal longevity supports the proposed ROS-mortality link.
loss on ROS levels. In agreement with the thermogenetic Dopamine transporter fumin (fmn) mutations cause sleep loss
approach, ROS accumulated in the gut and only in animals without affecting lifespan (Kume et al., 2005; Figures S2I). The
that experienced sustained sleep loss (Figure 3E). This supports guts of fmn mutants exhibited normal ROS levels, even after a
the idea that sleep deprivation itself causes ROS accumulation. month of severe sleep loss (Figure S2J). We have not yet uncov-
The fact that ROS levels increase with deprivation methods that ered the mechanisms that prevent ROS accumulation in this
either do (mechanical; Figure 3C) or do not (thermogenetic; Fig- case; regardless, we conclude that a normal lifespan with little
ure 1G, center and right) activate the sleep homeostat supports sleep is possible if ROS levels in the gut are kept low.
the notion that the physiological need for sleep is separable from
sleep drive (i.e., preventing sleepiness would not obviate the Cellular Evidence of Oxidative Stress upon Sleep
need for sleep). Deprivation
For the third deprivation method, we used loss-of-function ROS can destroy cellular macromolecules through oxidation
mutations or RNAi against known sleep regulators: Cyclin A (Evans et al., 2004; Gutteridge and Halliwell, 1990; Stadtman
(CycA) (Afonso et al., 2015; Rogulja and Young, 2012), Insomniac and Levine, 2003). As predicted from their high ROS levels,
(Inc) (Li et al., 2017; Pfeiffenberger and Allada, 2012; Stavropou- guts from sleep-deprived flies showed extensive evidence of
los and Young, 2011), Redeye (Rye), (Shi et al., 2014), and Sleep- oxidative stress (Figures 4A and 4B). Oxidation is harmful to
less (SSS) (Koh et al., 2008; Shi et al., 2014; Wu et al., 2010, DNA (Cadet and Wagner, 2013; Evans et al., 2004). In flies (Mad-
2014). As in the original studies, sleep was reduced with neuronal igan et al., 2002; Rogakou et al., 1999) and mammals (Rogakou
RNAi against Cyclin A (elav>CycARNAi) (Figures 3F and S2A) or et al., 1998), DNA double-strand breaks can be detected with an-
mutations in redeye (ryeT227M) (Figures 3H and S2B), sleepless tibodies that recognize histone H2A phosphorylation (gH2Av,
(sssD40) (Figures 3J and S2C), and insomniac (inc2) (Figures 3L gH2Ax in mammals) (Lake et al., 2013), a modification triggered
and S2D). In agreement with the other two deprivation methods, by the DNA damage response (Scully and Xie, 2013). This
ROS levels were elevated in the gut of these chronically deprived approach revealed widespread DNA damage in the gut (Figures
flies (Figures 3G, 3I, 3K, and 3M), reinforcing the idea that insuf- 4A and 4B). Oxidation also triggers adaptive pathways (Espi-
ficient sleep leads to ROS accumulation. nosa-Diez et al., 2015; Ma, 2013; Navarro-Yepes et al., 2014),
Lifespan was reduced by all of these genetic manipulations and there was an increase in stress granules (RNA-protein ag-
(Figures S2A–S2D), as previously described (Koh et al., 2008; gregates that halt translation and protect mRNA) (Buchan and
Rogulja and Young, 2012; Stavropoulos and Young, 2011). In Parker, 2009; Chen and Liu, 2017) and lysosomes (organelles
contrast to inc mutants, nervous system-specific depletion of that degrade damaged cell components) (Filomeni et al., 2015;
Inc was reported not to shorten lifespan (Hill et al., 2018; Stavro- Figures 4A and 4B). Sustained oxidation can ultimately cause
poulos and Young, 2011). These flies (elav>incRNAi) were indeed cells to die (Redza-Dutordoir and Averill-Bates, 2016); markers
not short lived (Figure S2G), but their sleep phenotype (Fig- of apoptosis and necrosis were observed throughout the gut
ure S2E) was milder than that of inc2 mutants (Figures 3L and (Figures 4A and 4B). Although these signs of cellular damage
S2D) and was not sustained (Figure S2F). Consistent with the fit the notion of oxidative stress where ROS are the inducers of
no-mortality phenotype, ROS levels were not elevated in ela- damage, there are other interpretations; in some contexts,
v>incRNAi flies (Figure S2H), suggesting that they have not accu- ROS can be downstream of cell-death-triggering signals (Kanda
mulated sufficient sleep debt. Sleep loss may not be the only et al., 2011; Pérez et al., 2017a; Zhang et al., 2009). Arguing that
cause of premature death in sleep mutants because the affected cellular damage and cell death are a consequence rather than
proteins function broadly. For example, Inc and SSS function at the cause of ROS increase, all oxidative stress markers ap-
larval neuromuscular synapses (Li et al., 2017; Wu et al., 2010), peared only after ROS began accumulating (Figures 4C and
and adult sss mutants exhibit motor neuron defects and lack co- 4D). When sleep deprivation was stopped and ROS levels grad-
ordination (Iyengar and Wu, 2014; Koh et al., 2008; Wu et al., ually decreased, markers of oxidative stress decreased as well
2010). Nonetheless, insufficient sleep and high ROS levels in (Figures S3A and S3B). Because the gut has high regenerative
the gut are phenotypes shared by these mutants. The general capacity (Amcheslavsky et al., 2009; Buchon et al., 2009b; Jiang
rule seems to be that ROS accumulate when sleep falls below et al., 2009; van der Flier and Clevers, 2009), the damaged cells
a certain threshold, which correlates with increased mortality. were likely replaced.
Figure 3. Other Methods of Sleep Suppression Also Lead to ROS Accumulation in the Gut
(A) Mechanical sleep deprivation varies in efficiency across individual flies. Each panel, 6 days of locomotor activity for an individual. Vertical ticks, 5-min lo-
comotor activity bins.
(B) Efficiency of mechanical deprivation varies across days in individuals (SL, sleep loss).
(C) Sleep rebound after 12 h of mechanical deprivation (SD) in isogenized (iso31) flies.
(D) Total amount of daily sleep in shaken (iso31 + SD) and non-shaken (iso31) flies for 6 days.
(E) Only in flies experiencing consistent (sustained) sleep loss, ROS accumulate in the gut. Day 7.
(F–M) Reduced sleep and high ROS levels in the gut of elav>CycARNAi flies (F and G) and ryeT227M (H and I), sssD40 (J and K), and inc2 (L and M) mutants.
Representative images. Scale bars, 200 mm. Mean and SEM.
For sample sizes and statistical analyses, see Table S1. See also Figure S2.
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The widespread cell death raised the possibility that the gut that ROS accumulation occurs regardless of the presence or
becomes structurally damaged and leaky, which we tested absence of compensatory sleep pressure.
with a commonly used method based on addition of unabsorb- Markers of DNA damage, stress granules, and cell death were
able blue dye to food. If the intestinal barrier is abnormally not observed after 2 days but could be clearly detected in the
permeable, such as in old flies, the entire body turns blue due small and large intestines after 5 days of sleep deprivation (Fig-
to an open circulatory system (Rera et al., 2011). With this ure 5D), again indicating that ROS are a cause rather than a
method, we could not detect a difference between deprived consequence of cellular damage. The weight of sleep-deprived
and non-deprived animals (Figure S3C), which suggests that mice dropped by 10% during the first 24 h of deprivation and
gut permeability was not majorly altered. We noticed that the then remained stable for the rest of the experiment (Figure S4E,
guts of sleep-deprived animals seemed to contain more food left). Food intake was comparable with the controls (Figure S4E,
(Figure S3C). This was due not to impaired excretion (Figure S3D, right), suggesting that the increase in ROS levels is not caused by
right) but to increased food intake (Figure S3D, left), consistent excessive eating. Despite the evidence mentioned earlier, food
with other sleep studies in humans and model organisms intake is not always increased during sleep restriction (Barf
(Baud et al., 2013; Everson et al., 1989; Greer et al., 2013; Koban et al., 2012; Caron and Stephenson, 2010). As would be consis-
et al., 2008; Markwald et al., 2013; Mavanji et al., 2013; Newman tent with another study (Koban et al., 2008), the severity and
et al., 2009; Rechtschaffen and Bergmann, 1995). We later pre- length of sleep deprivation that we imposed on mice may not
sent examples of sleep-deprived animals that do not eat more have been sufficient to appreciably change their appetite. Yet,
than controls but still have high ROS levels, which indicates these conditions were sufficient to induce ROS accumulation
that overeating is not the cause of ROS accumulation. in the gut. We conclude that the impact of sleep loss on the
gut is evolutionarily conserved and is separable from food
Sleep Deprivation Leads to ROS Accumulation and consumption.
Oxidative Stress in the Mammalian Gut
We tested the generalizability of our findings in a mammalian ROS Neutralization Extends the Survival of Sleep-
model. With gentle but continuous mechanical stimulation, Deprived Animals
C57BL/6J mice were deprived of sleep for up to 5 days (Figure 5). Our results reveal a correlation between sleep deprivation, ROS
In each repetition of the experiment, 5 mice were housed in a accumulation, and premature death. To test causality, we asked
deprivation setup from Pinnacle Technology (Hines et al., whether clearing ROS could extend survival without increasing
2013; Naidoo et al., 2014) where a slowly rotating bar caused sleep itself. Through food supplementation, we tested 53 individ-
them to move more frequently. As controls, 5 mice were housed ual compounds known to have antioxidant properties, identifying
in an identical setup but without the bar rotating. Sleep depriva- 11 that allowed a normal or near-normal lifespan (Figures 6A and
tion was confirmed with electroencephalogram (EEG)/electro- 6B). These included compounds that directly neutralize ROS
myogram (EMG) (Figure 5A), although the recordings were taken through electron donation (e.g., lipoic acid, quercetin, and
in singly housed mice and might be under-representing the 2,2,6,6-tetramethylpiperidine 1-oxyl [TEMPO]) as well as com-
extent of sleep loss in group-housed mice. Group housing pounds that boost the expression of endogenous antioxidant en-
caused animals to disturb each other (our observation): if one zymes (e.g., N-acetyl-L-cysteine [NAC] and sodium phenylbuty-
mouse was awake, it likely disrupted the sleep of other mice. rate [PBA]). Figure 6B shows examples of survival curves for
Internal organs from sleep-deprived and non-deprived mice three rescuing compounds: melatonin, lipoic acid, and NAD.
were processed in parallel; they were harvested, sliced, and The compounds that were able to rescue survival were effective
probed for ROS using DHE. Compared with the non-deprived at clearing ROS (Figure 6C and data not shown), unlike the non-
controls, sleep-deprived animals had elevated ROS levels spe- rescuing compounds we examined (Figures S5A and S5B).
cifically in the small and large intestines (Figures 5B and 5C). Rescue of survival was achieved without increasing sleep itself;
The increase in the small intestine was obvious after 2 days of all animals remained sleep deprived when supplemented with
deprivation, whereas the increase in the large intestine (colon) antioxidants (if anything, average sleep was reduced further)
was less obvious because of the seemingly higher basal ROS (Figure 6D). This was true even for melatonin, primarily known
levels (Figures 5B and 5C). There were no evident changes in for its effects on the mammalian circadian clock (Pfeffer et al.,
ROS in the other examined tissues, even after 5 days (Figures 2018). Melatonin does not seem to play a major role in controlling
5B and 5C). Sleep-depriving a different mouse strain (CBA/ circadian rhythmicity or sleep in flies (Hardeland and Poeggeler,
CaJ) produced a similar outcome, with ROS levels high in the 2003). A lesser known fact is that Melatonin is a potent antioxi-
small and large intestines but not in the brain or liver (Figures dant (Tan et al., 2015), produced at high levels in the vertebrate
S4A–S4C). When deprivation was stopped, mice engaged in gut (Bubenik, 2002). In flies, the rate-limiting enzyme in melatonin
rebound sleep (Figure S4D), supporting our earlier conclusion biosynthesis, arylalkylamine N-acetyltransferase, is highly
Figure 4. Sleep Deprivation Causes Oxidative Stress in the Gut

(A) Guts from sleep-deprived flies (green, blue) show high levels of oxidative stress markers. Some guts are outlined in white to help visualization.
(B) Quantification of oxidative stress markers in the guts of sleep-deprived (green, blue) and non-deprived flies.
(C) Oxidative stress markers are elevated only after 10 days of sleep deprivation, whereas ROS levels are already elevated on day 7.
(D) Quantification of ROS and oxidative stress markers in the guts of sleep-deprived (green, blue) and non-deprived flies. Mean and SEM.
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D
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expressed in the gut (Brodbeck et al., 1998). A recent study throughout the midgut in the most prevalent cell type, nutrient-
showed that circulating melatonin levels decrease with sleep absorbing enterocytes (the widespread pattern of oxidative
deprivation in mice (Gao et al., 2019). In agreement with our find- stress in the gut pointed to enterocytes as the most likely cell
ings, the authors found that melatonin supplementation attenu- type involved). The Gal4s used were mex1-Gal4 (Phillips and
ates mucosal injury and dysbiosis in the gut (Gao et al., 2019). Thomas, 2006; Figure 7C, left) and myo1A-Gal4 (Buchon et al.,
Based on our data, this effect of melatonin could be attributed 2013; Jiang et al., 2009; Figure S7A). mex1-Gal4 shows no
to its antioxidant properties. expression in the central nervous system (Figure 7C, left);
Together, these results draw a direct connection between myo1A-Gal4 has been reported not to be expressed in the brain
sleep deprivation-induced oxidative stress and mortality. If anti- (Takeishi et al., 2013), but with a more sensitive detection
oxidants can mean the difference between life and death for method (a brighter fluorescent reporter coupled with immuno-
sleep-deprived animals, one might predict life extension under labeling), we detected some expression there (Figure S7A; also
non-deprived conditions. Indeed, some groups have observed reported by Dai et al., 2019). Because of this, and because of a
beneficial effects of antioxidants on the healthspan and lifespan previous report suggesting oxidative stress in neurons of
of flies (Kang et al., 2002). However, supplementation with mela- sleep-deprived flies (Hill et al., 2018), we also attempted to
tonin, lipoic acid, NAD, or PBA, at doses that had rescuing ef- rescue survival using elav-Gal4, a driver that is expressed in
fects during sleep deprivation, did not affect the survival of most neurons and largely excluded from the gut (Figure 7C, right;
non-deprived animals (Figure S5C). This argues for a context- Chen et al., 2016). This allowed us to compare survival between
dependent value of antioxidants. sleep-deprived animals overexpressing antioxidant enzymes
throughout the gut (mex1-Gal4 and myo1A-Gal4) versus the ner-
Gut-Targeted Expression of Antioxidant Enzymes vous system (elav-Gal4).
Prevents Oxidative Damage and Extends the Survival of Gut-targeted overexpression of superoxide dismutase 1 or 2
Sleep-Deprived Animals (SOD1 and SOD2, respectively) or catalase (CAT) using mex1-
Compounds given through food easily reach gut cells, but we Gal4 (Figure 7D, top left) or myo1A-Gal4 (Figure S7B) rescued
could not exclude the possibility that they also reach other or- the survival of animals sleep-deprived with 50A07-LexA. The
gans and have some protective effects there. To directly test presence of myo1A-Gal4 (and, to a lesser extent, mex1-Gal4)
the role of the gut, we expressed antioxidant enzymes in this tis- somewhat reduced the long-term efficiency of sleep deprivation,
sue. This required two separate transgenic expression systems: independent of transgene expression (i.e., sleep levels gradually
one in neurons for thermogenetic sleep deprivation and the other increased over the lifetime in any animal that had myo1A-Gal4
in the gut for enzyme expression. The LexA/LexAop system (Lai [Figure S7C, top left] or mex1-Gal4 [Figure S7C, top right]) for
and Lee, 2006) is similar to the Gal4/UAS system (Brand and Per- reasons we do not understand. Regardless, during the initial
rimon, 1993). There are no available LexA drivers specific to the 11 days (the time it took for the sleep-deprived animals with no
gut, whereas such Gal4 drivers exist, so we searched for LexAs expression of transgenic antioxidant enzymes to die), average
to use for sleep deprivation. sleep loss was only weakly affected by myo1A-Gal4 (Figure S7C,
After screening through 150 sparsely expressed LexAs from bottom left) and not affected by mex1-Gal4 (Figure S7C, bottom
the FlyLight collection (Jenett et al., 2012; Pfeiffer et al., 2010), right). Moreover, loss of sleep throughout the lifespan was
we identified two (50A07- and 68A07-LexA) that allow long- comparable between sleep-deprived flies overexpressing anti-
term and consistent thermogenetic sleep deprivation (Figures oxidant enzymes with mex1-Gal4 and the sleep-deprived con-
7A, 7B, S6A, and S6B). The two lines show little expression in trols (Figure 7D, bottom left).
the gut (Figure S6C). Similar to what we observed using Gal4s, In contrast to gut-targeted overexpression, nervous system-
sleep deprivation with LexAs lead to premature death (Fig- targeted overexpression of antioxidant enzymes did not have a
ure S6D). 50A07-LexA caused nearly complete sleep loss and major rescuing effect, although lifespan was extended by several
precipitated death faster than 68A07-LexA, which caused days (Figure 7D, top right). This is consistent with the absence of
85% sleep loss (Figures 7B, S6B, and S6D). 50A07-LexA- noticeable ROS accumulation in the nervous system during
dependent deprivation was not accompanied by increased sleep deprivation (Figures 2B and 2C). The minor rescue ob-
food intake (Figure S6E), which was contrary to the Gal4-depen- tained with elav-Gal4 did not result from an increase in sleep
dent sleep deprivation but similar to the mouse experiments, re- (Figure 7D, bottom right) and could mean that there is some
inforcing the idea that excessive food consumption is not at the contribution of the nervous system to lethality, but it could also
root of sleep deprivation-triggered lethality. We combined result from the sparse elav-Gal4 expression in the gut (Chen
50A07-LexA with two independent Gal4 drivers expressed et al., 2016).
Figure 5. ROS Accumulate in the Mouse Gut upon Sleep Deprivation

(A) Rapid eye movement (REM) sleep, non-REM sleep, and wakefulness in sleep-deprived (SD) and non-deprived (non-SD) C57BL/6J mice, reported by
EEG/EMG.
(B) High ROS levels are seen in the small and large intestines after 2 days of sleep deprivation. Brain: h, hippocampus; c, cortex.
(C) Quantification of ROS levels in tissues from sleep-deprived and non-deprived mice.
(D) Markers of oxidative stress are elevated in the small and large intestine only after 5 days of sleep deprivation. Representative images. Scale bars: brain, 1 mm;
other tissues, 200 mm. Mean and SEM.
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With mex1- or myo1A-Gal4-driven overexpression of antioxi- other drives, such as hunger (Luquet et al., 2005), are separable
dant enzymes, ROS levels in the gut were brought down to base- from the physiological need they represent.
line (SOD1 or SOD2 overexpression) or even seemingly below How does sleep deprivation lead to ROS accumulation? Why
(CAT overexpression) (Figures 7E, left, and S7D and S7F, left, does this take place in the gut? It might be that ROS clearance is
and S7G). DNA damage and apoptosis were no longer detect- a daily sleep function, but it is also possible that sleep depriva-
able (Figures 7E, center and right, and S7E and S7F, center tion generates unique adverse conditions that lead to ROS accu-
and right, and S7H), directly demonstrating that ROS are the mulation. ROS levels may result from increased production,
cause of cellular damage in the gut of sleep-deprived flies. The decreased elimination, or both. Prolonged wakefulness could
efficacy of the mex1- or myo1A-Gal4-dependent rescue sug- affect the gut directly, but ROS accumulation could also be a
gests that enterocytes are the major source of ROS in the gut consequence of signaling from the nervous system or other
upon sleep deprivation. With elav-Gal4-driven overexpression tissues.
of antioxidant enzymes, ROS levels in the gut remained high (Fig- The vast majority of ROS (90%) are generated during mito-
ure S6F). When flies were sleep-deprived with the other LexA line chondrial oxygen-dependent ATP synthesis (Balaban et al.,
(68A07-LexA), and SOD1, SOD2, or CAT were overexpressed in 2005), making this organelle an obvious candidate for the
the gut using myo1A-Gal4, lifespan was also extended (Figures relevant ROS source. Metabolism increases during sleep disrup-
S6G and S6H). The results of these rescue experiments reinforce tion (Barf et al., 2012; Bonnet et al., 1991; Caron and Stephen-
our conclusion that the gut is the main source of lethal ROS dur- son, 2010; Everson et al., 1989; Everson and Wehr, 1993; Stahl
ing severe sleep loss (Figure 7F). et al., 2017; Valenti et al., 2017), and metabolic regulation may
have been an original sleep function. The concentration of atmo-
spheric oxygen changed significantly several times in Earth’s
DISCUSSION history, which is thought to have led to eukaryotic and multicel-
lular life (El Albani et al., 2010; Sessions et al., 2009). Oxygen use
Our study uncovered a cause of death upon experimental sleep increased the efficiency of ATP production (Stamati et al., 2011)
deprivation. One caveat of this and other sleep deprivation but came with the price of having to neutralize reactive and
studies in animal models is the accompanying overall increase potentially toxic oxygen derivatives (Fischer et al., 2016; Taverne
in physical activity that could contribute to ROS production et al., 2018). Entering a resting state characterized by reduced
through higher energy expenditure. We do not think that this is metabolism could temper ROS production by decreasing the
a major factor behind the observed phenomena because sleep rate of redox reactions. The gut appeared early in evolution
is not merely a period of immobility. Metabolic changes associ- (Stainier, 2005) and could be uniquely sensitive to metabolic per-
ated with normal daily sleep-wake cycles are separable from turbations. It is not clear, however, whether the gut has any spe-
changes in locomotion (Bonnet et al., 1991; Stahl et al., 2017), cial energetic demands (Aiello and Wheeler, 1995) that explain
and increased locomotion by itself does not seem to contribute the increase in ROS seen there. Moreover, increasing mitochon-
significantly to the increased metabolic rate and energy expendi- drial activity in the gut can actually reduce ROS levels and exten-
ture during experimental sleep deprivation (Barf et al., 2012; dlifespan (Rera et al., 2011). Another potential source of ROS is
Bergmann et al., 1989; Caron and Stephenson, 2010). Addition- the endoplasmic reticulum (ER), where ROS are routinely made
ally, intense physical activity increases the levels of ROS in mus- as byproducts of oxidative protein folding (Shimizu and Hender-
cles (Powers and Jackson, 2008), but we did not detect changes shot, 2009). ER stress (the accumulation of misfolded or
in this tissue, suggesting that our observations do not stem sim- unfolded proteins) can lead to oxidative stress, as increased de-
ply from physical exhaustion. The second caveat is that we were mand for protein folding leads to greater ROS production; ER
able to study survival and demonstrate rescue only with thermo- stress also induces mitochondria to produce more ROS (Cao
genetic deprivation because only this approach allowed consis- and Kaufman, 2014; Santos et al., 2009; Zeeshan et al., 2016).
tent and sustained sleep suppression without any obvious side Previous studies found that sleep-deprived brains show evi-
effects. Regardless, ROS accumulation was observed with dence of unfolded protein response (Brown and Naidoo, 2010;
different methods of suppressing sleep and at different temper- Brown et al., 2017; Hakim et al., 2015; Naidoo et al., 2005,
atures. Two of these methods (thermogenetics and mutations in 2007; Shaw et al., 2000), which might in turn trigger a response
sleep genes) suppress sleep without triggering (or weakly trig- in the gut (Berendzen et al., 2016; Schinzel and Dillin, 2015; Tay-
gering) an increase in homeostatic sleep pressure, but mechan- lor and Dillin, 2013; Zhang et al., 2018). Alternatively, sleep depri-
ical agitation robustly engages the homeostat. From this, we vation could directly induce ER stress in the gut. In both sce-
conclude that the physiological need for sleep can be separated narios, ER stress in the gut may lead to oxidative stress in this
from the compensatory sleep drive, consistent with the fact that tissue.
Figure 6. Clearance of ROS Prevents Death of Thermogenetically Sleep-Deprived Flies

(A) Individual survival. Sleep-deprived flies (11H05>TrpA1, 60D04>TrpA1) die earlier than the parental controls (11H05-Gal4, 60D04-Gal4, UAS-TrpA1). Solvents
(ethanol, DMSO) do not affect survival. Supplementation with antioxidants (melatonin–vitamin C) extends the survival of sleep-deprived flies. Mean and SEM.
(B) Example survival curves for three rescuing compounds. Error bars are omitted for clarity.
(C) ROS are cleared from the gut by the compounds that rescue survival. Day 10. Representative images. Scale bar, 200 mm. Mean and SEM.
(D) Sleep is not promoted by the rescuing antioxidants. Mean and SEM.
Survival experiments were performed 2–3 times. For sample sizes and statistical analyses, see Table S1. See also Figure S5.
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A B
E F
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Gut homeostasis depends on physiological levels of locally Why could we and others (Cirelli, 2006; Cirelli et al., 1999;
-produced ROS (Campbell and Colgan, 2019; Pérez et al., D’Almeida et al., 1997; Eiland et al., 2002; Gopalakrishnan
2017b) that regulate stem cell proliferation (Buchon et al., et al., 2004; Hipólide et al., 2002) not detect any significant
2009a; Hochmuth et al., 2011; Jones et al., 2013; Patel et al., oxidative damage in sleep-deprived brains? The nervous sys-
2019; Wang et al., 2014; Xu et al., 2017) and function in intestinal tem has high energetic demands, and neuronal ATP production
immunity (Buchon et al., 2009a; Ha et al., 2005, 2009; Jones et al., is almost certain to rise during prolonged wakefulness, which is
2013; Jones and Neish, 2017; Kim and Lee, 2014). A significant expected to elevate ROS levels. The lack of damage, as pro-
fraction of intestinal ROS are made through the action of the posed previously (Cirelli, 2006), might be at least partially ex-
conserved enzymes Nox and Duox (Aviello and Knaus, 2018; plained by efficient activation of antioxidant and cytoprotective
Ha et al., 2005; Jones et al., 2013). Nox is hyperactivated by dys- mechanisms in sleep-deprived brains (Brown et al., 2017; Cirelli
biosis (Iatsenko et al., 2018), alteration of the gut microbial profile et al., 2004, 2005b, 2006; Cirelli and Tononi, 2000; Hill et al.,
(Levy et al., 2017). This might be relevant because sleep depriva- 2018; Jones et al., 2008; Naidoo et al., 2005; Nikonova et al.,
tion reportedly affects gut microbiota (Benedict et al., 2016; El 2010; Shaw et al., 2000; Terao et al., 2003, 2006). Oxidation reg-
Aidy et al., 2019; Gao et al., 2019; Ma et al., 2019; Poroyko ulates the activity of a small group of sleep-regulating neurons in
et al., 2016), though conflicting evidence exists (Zhang et al., flies (Kempf et al., 2019), but it is likely critical to keep oxidation
2017). Intestinal Nox is also activated by stressors other than dys- generally low in neurons because they cannot be replaced, in
biosis (Patel et al., 2019), and sleep deprivation could be one such contrast to gut cells which undergo constant turnover (acceler-
stressor. The gut has the ability to receive distress signals from ated by tissue damage)(Amcheslavsky et al., 2009; Buchon et
other tissues (Berendzen et al., 2016; Liu and Jin, 2017; Ulgherait al., 2009; Jiang et al., 2009; Micchelli and Perrimon, 2006; Mi-
et al., 2014; Zhang et al., 2018) and initiate protective responses. guel-Aliaga et al., 2018; Ohlstein and Spradling, 2006; van der
For example, sterile external wounds can cause a ROS increase Flier and Clevers, 2009). Because the gut is plastic in terms of
in the fly gut, which promotes enterocyte turnover and improves proliferation, recovery from oxidative damage when deprivation
survival (Takeishi et al., 2013). ROS levels could similarly start is stopped may rely mostly on turning cells over rather than re-
increasing as a protective response in the gut during periods of pairing them. It will be informative to test whether sleep pro-
insufficient sleep. Under natural conditions, animals likely fall motes active repair of cellular damage in the gut, similar to
asleep before ROS reach dangerously high levels. We consider the way it facilitates DNA damage repair in neurons (Bellesi
this scenario possible because antioxidant compounds some- et al., 2016; Zada et al., 2019).
times accelerated death of sleep-deprived animals (if given too Regardless of how or why they accumulate during insufficient
early or at doses that were too high, data not shown). sleep, excessive intestinal ROS are lethal, consistent with previ-
ROS could accumulate not only through increased production ous reports (Ha et al., 2009; Iatsenko et al., 2018; Lee et al.,
but decreased clearance. Inadequate neutralization coupled 2013). The widespread cellular damage we observed was not
with constant production in the gut (Campbell and Colgan, accompanied by the breakdown of the intestinal barrier seen in
2019; Jones and Neish, 2017; Pérez et al., 2017b) could explain older animals (Rera et al., 2011, 2012), suggesting that sleep
gradual accumulation of these molecules. Nrf2 (CnC in flies) is a deprivation is not simply accelerated aging. To strengthen this
conserved master antioxidant regulator whose basal activity is conclusion, other hallmarks of aging, such as intestinal dysplasia
higher in the fly gut than in other tissues (Sykiotis and Bohmann, (Biteau et al., 2010; Jasper, 2020), should be examined, espe-
2008). A transcription factor, Nrf2 becomes stabilized and trans- cially given that ROS regulate intestinal stem cell proliferation
locates into the nucleus only under oxidative conditions to (Biteau et al., 2008; Buchon et al., 2009a; Choi et al., 2008; Hoch-
induce expression of antioxidant and cytoprotective genes muth et al., 2011; Jones et al., 2013; Patel et al., 2019; Wang
(Ma, 2013; Sykiotis and Bohmann, 2008; Tonelli et al., 2018). A et al., 2014; Xu et al., 2017). Oxidative damage might interfere
defect in this mechanism could result in gut-biased ROS accu- with the absorptive function of the gut, and the increased feeding
mulation. At least several antioxidant compounds that rescued seen during sleep loss (Baud et al., 2013; Everson et al., 1989;
the survival of sleep-deprived flies can act on Nrf2 (Rochette Koban et al., 2008; Mavanji et al., 2013; Newman et al., 2009; Re-
et al., 2013; Vriend and Reiter, 2015), supporting the idea of po- chtschaffen and Bergmann, 1995) could be an attempt to
tential Nrf2 dysfunction during sleep loss. compensate for nutrient deficiency. In agreement with this
Figure 7. Overexpression of Antioxidant Enzymes in the Gut Prevents Death of Thermogenetically Sleep-Deprived Flies
(A) Expression of 50A07-LexA in the nervous system, reported by mCD8::GFP
(B) Sleep across 3 days. Raising the temperature to 29 C (orange) causes sleep loss in 50A07>TrpA1 flies (these experiments were done in darkness because the
efficiency of deprivation was greater than in light-dark cycles). Mean and SEM. Right: daily sleep in controls and experimental flies across the lifespan. Mean
and SEM.
(C) Expression pattern of mex1-Gal4 (left) and elav-Gal4 (right) in the gut and the central nervous system, visualized with nls-mCherry. Scale bars, 200 mm.
(D) Top: survival of sleep-deprived flies overexpressing antioxidant enzymes (SOD1, SOD2, or CAT) in the gut (mex1-Gal4, left) or the nervous system (elav-Gal4,
right). Dot plot, survival for individuals flies. Mean and SEM. Curves, population survival. Error bars are omitted for clarity. Bottom: daily sleep of sleep-deprived
flies overexpressing antioxidant enzymes (SOD1, SOD2, or CAT) in the gut (left) or the nervous system (right).
(E) Overexpression of SOD1 in the gut of SD flies (50A07>TrpA1, mex1>SOD1) prevents ROS accumulation and oxidative stress. Representative images. Scale
bars, 200 mm. Guts are outlined for visualization. Survival experiments were performed 3 times.
(F) Graphic summary. The model was created with BioRender.
For sample sizes and statistical analyses, see Table S1. See also Figures S6 and S7.
Cell 181, 1–22, June 11, 2020 15

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idea, a high-calorie diet can delay death of sleep-deprived rats B Drosophila melanogaster
(Everson and Wehr, 1993). B Mice
Accumulated intestinal ROS might have systemic effects, for d METHOD DETAILS
example by altering the gut microbial profile through direct B Drosophila melanogaster
bactericidal action or through modulation of gut immunity B Mice
(Campbell and Colgan, 2019; Ha et al., 2005; Yardeni et al., d QUANTIFICATION AND STATISTICAL ANALYSIS
2019). Since microbiota regulate health and lifespan (Clark
et al., 2015; Clark and Walker, 2018; Heintz and Mair, 2014; SUPPLEMENTAL INFORMATION
Smith et al., 2017; Thaiss et al., 2016), this might be a factor in
early mortality. Intestinal ROS could also trigger a systemic in- Supplemental Information can be found online at https://doi.org/10.1016/j.
cell.2020.04.049.
flammatory response independently of microbiota (Wu et al.,
A video abstract is available at https://doi.org/10.1016/j.cell.2020.04.
2012). Because ROS are important signaling molecules at phys- 049#mmc2.
iological levels (Holmström and Finkel, 2014; Jones, 2006; Li
et al., 2018; Lim et al., 2014; Mittler, 2017; Oswald et al., 2018; ACKNOWLEDGMENTS
Owusu-Ansah and Banerjee, 2009; Schieber and Chandel,
2014), lethality could also result from disruption of the normal We thank the Rogulja and Crickmore labs, Jesse Goldberg, Gary Yellen, and
ROS-dependent processes. Charles Weitz for helpful discussions; Stephen Zhang and Bryan Song for
Severe conditions were presented here, but mild deprivation the custom MATLAB programs for sleep and survival analyses; Manon Bon-
jour, Felipe Cybis Pereira, Elin Hu, Peri Matatia, Victor Nan, Nedah Nemati,
also leads to ROS accumulation in the gut, albeit slower (data
Aleksandra Prochera, Emory Sabatini, and Fergus Wade for technical assis-
not shown). This means that our results are likely relevant for tance; and Anaı̈s Aulas for guidance and reagents to examine stress granules.
chronic insufficient sleep in humans, especially considering For flies, we thank Claire Thomas (mex1-Gal4), Norbert Perrimon (myo1A-
that, among the many pathologies associated with sleep restric- Gal4), Rob Jackson (fumin mutants), Nicholas Stavropoulos (elav-Gal4;
tion (Chattu et al., 2018), gastrointestinal diseases feature prom- UAS-Dcr2, UAS-incRNAi, and insomniac mutants), and Amita Sehgal (redeye
inently (Ali et al., 2013; Khanijow et al., 2015; Parekh et al., 2018) and sleepless mutants). D.R. is a New York Stem Cell Foundation-Robertson
and that several gastrointestinal disorders are characterized by Investigator. This work was supported by grants from the New York Stem
Cell Foundation and the Pew Charitable Trust (to D.R.) and the NIH (R73
abnormal ROS levels (Bhattacharyya et al., 2014; Campbell
NSO72030 to M.G.); EMBO long-term fellowship ALTF 1345-2015, a Fonda-
and Colgan, 2019; Pérez et al., 2017b). The risk of colon cancer tion Bettencourt Schueller fellowship, and Lefler postdoctoral fellowship (to
is elevated by insufficient or untimely sleep (Schernhammer A.V.); a Brooks postdoctoral fellowship (to Y.K.D.); and a Life Sciences
et al., 2003; Thompson et al., 2011); intestinal ROS might be research fellowship (to E.A.P.).
tumorigenic through several mechanisms (Bhattacharyya et al.,
2014), such as damaging DNA (Evans et al., 2004; Markowitz AUTHOR CONTRIBUTIONS
and Bertagnolli, 2009) or causing inflammation (Aviello and
Knaus, 2017; Lasry et al., 2016). Inflammation is frequently A.V. and D.R. conceived and initiated the study. A.V., Y.K.D., K.N., E.A.P., C.L.,
and D.R. designed the experiments and analyzed the data. A.V., Y.K.D., K.N.,
observed with insufficient sleep (Irwin, 2019; Mullington et al.,
E.A.P., and C.L. performed the experiments. A.V., Y.K.D., and D.R. wrote the
2010) and is increasingly recognized as a driver of disease manuscript with input from all authors.
(Chung et al., 2009; Elinav et al., 2013; Gao and Hong, 2008;
Libby, 2007; Rea et al., 2018). Inflammation could also trigger DECLARATION OF INTERESTS
ROS production by immune cells (Mittal et al., 2014), leading to
oxidation of other organs. With a longer deprivation protocol A patent to A.V. and D.R. is pending (‘‘A method for treating damage induced
than we describe in mice, oxidative DNA damage was indeed by sleep deprivation’’).
seen, in addition to the small intestine, in the lung and the liver
Received: May 15, 2019
of rats (Everson et al., 2014). We recognize that what kills
Revised: January 15, 2020
extremely sleep-deprived animals might not reflect what sleep Accepted: April 24, 2020
does daily. Regardless, our findings demystify the observation Published: June 4, 2020
that extreme sleep loss can cause death and show that it is
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER

Fly Stocks
11H05-Gal4 BDSC (Bloomington Drosophila Stock RRID: BDSC_45016
Center)
60D04-Gal4 BDSC RRID: BDSC_45356
UAS-mCD8::GFP Gift from Michael Crickmore N/A
UAS-TrpA1 BDSC RRID: BDSC_26263
iso31 Ryder et al., 2004 N/A
elav-Gal4; UAS-Dcr2 Gift from Nicholas Stavropoulos N/A
UAS-CycARNAi NIG (National Institute of Genetics) 5940R-1; FBgn0000404
iso31-AS Gift from Amita Sehgal N/A
ryeT227M Gift from Amita Sehgal N/A
sssD40 Gift from Amita Sehgal N/A
w1118-NS Gift from Nicholas Stavropoulos N/A
2
inc Gift from Nicholas Stavropoulos N/A
50A07-LexA BDSC RRID: BDSC_53538
68A07-LexA BDSC RRID: BDSC_61586
LexAop-mCD8::GFP BDSC RRID: BDSC_32203
LexAop-TrpA1 Burke et al., 2012 N/A
(via Michael Crickmore)
mex1-Gal4 (Chromosome X insertion) Gift from Claire Thomas N/A
elav-Gal4 BDSC RRID: BDSC_458
myo1A-Gal4 Gift from Norbert Perrimon N/A
UAS-nls-mCherry BDSC RRID: BDSC_38424
UAS-SOD1 BDSC RRID: BDSC_33605
UAS-SOD2 BDSC RRID: BDSC_24494
UAS-CAT BDSC RRID: BDSC_24621
UAS-RedStinger Gift from Michael Crickmore N/A
esg-Gal4 BDSC RRID: BDSC_26816
UAS-incRNAi Gift from Nicholas Stavropoulos N/A
w1118-RJ Gift from Rob Jackson N/A
fmnrec19 Gift from Rob Jackson N/A
LexAop-RedStinger Gift from Michael Crickmore N/A
Antibodies
chicken anti-GFP Aves Cat# GFP-1020; RRID: AB_10000240
mouse anti-Bruchpilot DHSB (Developmental Studies Cat# nc82; RRID: AB_2314866
Hybridoma Bank)
mouse anti-gH2Av DHSB Cat# UNC93-5.2.1; RRID: AB_2618077
mouse anti-FMR1 DHSB Cat# anti-dFMR1, 5A11; RRID: AB_528252
rabbit anti-cleaved Caspase 3 Cell Signaling Technology Cat# 9661; RRID: AB_2341188
mouse anti-gH2Ax Cell Signaling Technology Cat# 2577; RRID: AB_2118010
mouse anti-TIA1 Santa-Cruz Cat# sc-166247; RRID: AB_2201545
rabbit anti-dsRed Takara Bio Cat# 632496; RRID: AB_10013483
Alexa Fluor 488 donkey anti-mouse Molecular Probes Cat# A-21202; RRID: AB_141607
Alexa Fluor 488 donkey anti-rabbit Molecular Probes Cat# A-21206; RRID: AB_2535792
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Continued
Alexa Fluor 555 donkey anti-rabbit Molecular Probes Cat# A-31572; RRID: AB_162543
Alexa Fluor 568 donkey anti-mouse Thermo Fisher Scientific Cat# A10037; RRID: AB_2534013
Alexa Fluor 647 donkey anti-mouse Molecular Probes Cat# A-31571; RRID: AB_162542
Alexa Fluor 488 donkey donkey anti- Jackson ImmunoResearch Labs Cat# 703-545-155; RRID: AB_2340375
chicken
Antioxidant Compounds
Melatonin Sigma-Aldrich Cat# M5250; CAS: 73-31-4
Lipoic acid Sigma-Aldrich Cat# T5625; CAS: 1077-28-7
Nicotinamide adenine dinucleotide (NAD) Sigma-Aldrich Cat# N0632; CAS: 20111-18-6
Coenzyme-Q10 Sigma-Aldrich Cat# C9538; CAS: 303-98-0
5,10,15,20-Tetrakis(1-methyl-4-pyridinio) Sigma-Aldrich Cat# 323497; CAS: 36951-72-1
porphyrin tetra(p-toluenesulfonate) (TMPyP)
Resveratrol VWR CAS: 501-36-0
2,2,6,6-Tetramethylpiperidine 1-oxyl (TEMPO) Sigma-Aldrich Cat# 176141; CAS: 2226-96-2
Quercetin Sigma-Aldrich Cat# Q4951; CAS: 117-39-5
Sodium phenylbutyrate (PBA) Sigma-Aldrich Cat# SML0309; CAS: 1716-12-7
N-Acetyl-L-cysteine (NAC) Sigma-Aldrich Cat# A7250; CAS: 616-91-1
Vitamin C Sigma-Aldrich Cat# A5960; CAS: 50-81-7
Ebselen Sigma-Aldrich Cat# E3520; CAS: 60940-34-3
Glutathione Sigma-Aldrich Cat# PHR1359; CAS: 70-18-8
Methylene blue Sigma-Aldrich Cat# 28514; CAS: 122965-43-9
Other
Agar Moorhead Cat# 41084
BSA Gemini Cat# 700-101P; CAS: 9048-46-8
C&B metabond quick adhesive cement Parkell Cat# S380
system
Cornmeal (Degermed yellow) Bunge Cat# CCM 250
DAPI Fluoromount-G SouthernBiotech Cat# 0100-20
DHE Sigma-Aldrich Cat# 37291; CAS: 104821-25-2
DMSO VWR Cat# 97063-136; CAS: 67-68-5
Ethanol (pure) Koptec Cat# V1001; CAS: 64-17-5
Hydrophobic Barrier Pen Vector Laboratories Cat# H-4000; RRID: AB_2336517
KPL 10X Dulbeco’s PBS SeraCare Cat# 5460-0030
Loctite 404 Instant Adhesive Fisher Scientific Cat# NC0619400
LysoTracker Red Invitrogen Cat# L12492
Microscope coverslips Electron Microscopy Sciences Cat# 72230-01
Microscope coverslips VWR Cat# 48393-251
Microscope glass slides Super Frost Plus VWR Cat# 48311-703
Microscope glass slides Electron Microscopy Sciences Cat# 63421-10
Normal goat serum Jackson ImmunoResearch Cat# 005-000-121; RRID: AB_2336990
O.C.T (Tissue-Tek) Electron Microscopy Sciences Cat# 62550-01
PFA (16%) Electron Microscopy Sciences Cat# 15710; CAS: 30525-89-4
Prolong Gold antifade reagent Invitrogen Cat# 1942345
Proprionic acid Fisher Cat# A258-500; CAS: 79-09-4
Sugar (premium pure cane granulated) Domino
Schneider’s medium GIBCO Cat# 21720-001
SYTOX Green Invitrogen Cat# S7020
Tegosept Genesee Cat# 20-258
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Triton X-100 Amresco Cat# M143; CAS: 9002-93-1
Vectaschield with DAPI Vector Laboratories Cat# H-1200; RRID: AB_2336790
Yeast torula MP Biomedicals Cat# 903085
RESOURCE AVAILABILITY
Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dragana
Rogulja (dragana_rogulja@hms.harvard.edu).
Materials Availability
This study did not generate new unique reagents.
Data and Code Availability

Data are available from the corresponding author on request, and sleep analysis codes are available on github at https://github.com/
CrickmoreRoguljaLabs/SleepAnalysis.
EXPERIMENTAL MODELS AND SUBJECT DETAILS
Drosophila melanogaster
All Drosophila stocks used in this study are listed in the key resources table. Mutant flies were compared to wild-type controls of the
same genetic background. Flies were maintained on cornmeal-agar medium in 12 hours light:12 hours dark cycles (12:12 LD), unless
indicated otherwise. The cornmeal-agar medium was composed of agar (7.3%, Spectrum), yeast (12.7%, MP Biomedicals), sugar
(56%, Domino), and cornmeal (24%, Bunge). To make 4 L of cornmeal-agar medium, water temperature was brought to 50 C. Agar,
yeast, sugar, and cornmeal were added slowly, with constant stirring to prevent clumping. Temperature was increased until bubbles
formed (around 97 C), after which the heat was turned off. Once temperature dropped to 75 C, propionic acid (100 ml) and Tego-
sept + ethanol (95.2 g in 367.2 ml) were added and water was adjusted to compensate for evaporation. The mix was left for 15 minutes
before pouring into vials, to allow homogenization. All experiments were performed using adult males and carried out in incubators
purchased from Tritech Research Inc. (standard DigiTherm model) and Percival Scientific Inc. (DR36VL model).
Mice
2-month-old male C57BL/6J (JAX 000664) and CBA/CaJ (JAX 000654) mice were housed according to standard protocols approved
by the Harvard University Standing Committee on Animal Care in accordance with federal guidelines. On arrival to our animal facility,
mice were housed in pathogen-free cages (max 5 per cage) under 12:12 LD cycles, at 23 C and 50% humidity. They were given stan-
dard Pico Lab Rodent Diet 20 # 5053, and water.
METHOD DETAILS
Drosophila melanogaster
Locomotor activity and sleep monitoring
Locomotor activity and sleep were tracked using commercially available Drosophila Activity Monitors (DAMs, TriKinetics). Individual
males were placed into 65 mm-long glass tubes (TriKinetics) containing 20 mm of cornmeal-agar food. Locomotor activity data
were collected from the DAM System (TriKinetics) using DAM5M monitors, unless indicated otherwise. Each DAM5M monitor mea-
sures locomotor activity of 32 flies. 4 independent infrared beams are positioned across each tube. As a fly walks back and forth, it
interrupts the infrared beams, leaving a record of when and where it moved. Total movement counts across the 4 beams were re-
corded and summed every minute. In experiments using mechanical sleep deprivation and RNAi against Cyclin A (CycARNAi),
DAM2 activity monitors were used. These monitors also measure activity of up to 32 individual flies, but there is only 1 infrared
beam to monitor movement, potentially under-reporting total movements. Data from DAM5M or DAM2 monitors were then analyzed
using a custom MATLAB software (available on github at https://github.com/CrickmoreRoguljaLabs/SleepAnalysis). A sleep episode
was defined as inactivity lasting at least five minutes, based on the original characterization of fly sleep (Andretic and Shaw, 2005; Nitz
et al., 2002; Shaw et al., 2000).
For each genotype/condition, average daily sleep was calculated for the entire lifespan, unless indicated otherwise. Locomotor
activity data were collected at either 25 C (redeye (ryeT227M), sleepless (sssD40), insomniac (inc2) and fuminrec19 (fmn) mutants, insom-
niac RNAi (incRNAi) and mechanical sleep deprivation); 27 C (RNAi against Cyclin A (CycARNAi)), or 29 C (Thermogenetic deprivation).
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Glass tubes were replaced every 2-3 days to avoid food desiccation. For antioxidant feeding assays, the antioxidant compounds
were added to food at concentrations listed below.
Sleep deprivation
Thermogenetic sleep deprivation was performed using the Gal4/UAS and LexA/LexAop systems. Flies were raised in 12:12 LD cycles
at 21 C until day 4-5 post-eclosion, after which males were loaded into TriKinetics glass tubes and placed in DAM5M monitors. After
one day of baseline sleep recording at 21 C, temperature was raised to constant 29 C, which triggered sleep deprivation. Deprivation
was done in 12:12 LD cycles, except for the experiments involving the LexA/LexAop system – these were done in constant darkness
because deprivation using the LexA lines was more consistent in the absence of light.
Mechanical sleep deprivation was performed at 25 C using a multi-tubes vortexer (VWR) modified by TriKinetics to house DAM2
activity monitors. Flies were raised at 25 C (12:12 LD) until day 4-5 post-eclosion, when males were loaded into tubes. After a day of
baseline sleep recording, the multi-tubes vortexer delivered 2 s-long vibrations at random intervals centered around 60 s (±30 s). The
intensity of the vortexer was set to 7.
Sleep rebound was measured after one night (12 hours, ZT12-ZT24) of mechanical sleep deprivation. Rebound sleep (D sleep) was
calculated for each fly by subtracting the amount of sleep on the morning before deprivation (ZT0-ZT4) from the amount of sleep on
the morning (ZT0-ZT4) following the deprivation.
Lifespan assay
Survival was measured on cornmeal-agar medium. In all survival experiments, males were transferred to fresh vials or tubes every 2-
3 days, without using CO2. Each survival experiment was performed at least twice. For thermogenetic sleep-deprivation experi-
ments, crosses were kept at 21 C (12:12 LD). Newly eclosed flies were collected every two days and kept at 21 C (12:12 LD) until
day 4-5 post-eclosion. Males were then transferred to 29 C under either 12:12 LD cycles (for deprivation experiments using Gal4/
UAS) or to constant darkness (for deprivation experiments using LexA/LexAop). For CyclinA RNAi (CycARNAi) experiments, crosses
were kept at 25 C (12:12 LD). Newly eclosed males (day 0-1 post-eclosion) were transferred to 27 C (12:12 LD), as in the original
publication. redeye (ryeT227M), sleepless (sssD40), insomniac (inc2) and fuminrec19 (fmn) mutants, as well as crosses of insomniac
RNAi (incRNAi) were maintained at 25 C (LD 12:12). Newly eclosed males (0-1 days post-eclosion) were collected and kept at
25 C (LD 12:12) until death. For genetic rescue of LexA-dependent thermogenetic sleep deprivation and for redeye (ryeT227M) and
sleepless (sssD40) mutants, single flies were loaded into TriKinetics glass tubes and placed in TriKinetics DAM2 or DAM5M monitors
to assess survival (only locomotor data from DAM5M monitors were used for calculating sleep amount, due to better resolution.
Death was determined on the day when locomotor activity stopped permanently, as evident by locomotor activity recordings. At least
15 males per genotype per experiment were tested. In all the other survival experiments, males were placed into standard fly vials. At
least 3 vials per genotype per experiment were used, with 10-20 animals per vial. We initially tested if survival depended on the num-
ber of animals per vial (10 versus 15 versus 20) and on whether the flies were grouped in vials or placed individually in TriKinetics glass
tubes, and did not observe any difference (data not shown). Survival with antioxidant feeding was measured as described above, but
with single flies in TriKinetics glass tubes. At least 10 males per genotype per condition per experiment were tested. Flies were
counted as dead if they failed to move (e.g., leg movements) in response to taps on the side of the vials or tubes.
Antioxidant Feeding
Individual antioxidant compounds were diluted in the appropriate solvent (H2O, DMSO or EtOH) and added to the cornmeal-agar
food. Animals were put on this food after 6 complete days at 29 C (Day 12 post-eclosion, Figures 6 and S5), and transferred to fresh
tubes every 2-3 days (without using CO2) to avoid compound degradation and food desiccation. Antioxidants were used at the
following final concentrations: Coenzyme-Q10, 1 mM (in EtOH); Ebselen, 1 mM (in DMSO); Glutathione, 5 mM (in DMSO); Lipoic
acid, 2 mM (in EtOH); Melatonin, 100 mg/ml (in EtOH); Methylene blue, 5 mM (in H2O); NAC, 20 mM (in H2O); NAD, 10 mM (in
DMSO); PBA 20 mM (in H2O); Quercetin 5 mM (in DMSO); Resveratrol, 10 mM (in DMSO); TEMPO, 10 mM (in DMSO); TMPyP,
10 mM (in H2O); Vitamin C, 40 mM (in H2O).
Food intake measurement in thermogenetically sleep-deprived flies
After 5 days (Gal4-dependent sleep deprivation) or 4 days (LexA-dependent sleep deprivation) at 29 C, males were transferred from
standard cornmeal-agar medium to otherwise identical media containing 2% (wt/vol) FD&C Blue #1. 24 hours later, feeding on blue
food was interrupted by freezing the vials at 20 C. Frozen flies were transferred to Eppendorf tubes (5 males per tube), decapitated
(to prevent the eye pigment from interfering with the subsequent measurement), and homogenized with a motorized pestle (VWR) in
50 mL 1X-PBS + 1% Triton X-100. Samples were centrifuged to clear the debris and the absorbance of the supernatant was measured
at 660 nm (A660) on a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). Flies fed regular non-blue food were used as
controls, and their A660 values were subtracted from experimental values. Serial dilutions of the dye were used to generate a stan-
dard curve. The dye concentration of each sample was determined using the linear fit of the standard curve. For each genotype, a
minimum of 15 males were tested.
Smurf assay
FD&C blue #1 dye was added at a concentration of 2.5% (wt/vol) to standard cornmeal-agar medium. Males were maintained on
dyed medium from the first day of sleep deprivation until death. Smurf flies are characterized by blue coloration present outside
the digestive tract. We did not observe any Smurf phenotypes in sleep-deprived flies : the blue coloration stayed restricted to the gut.
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Gut Immunohistochemistry
Adult males were anesthetized on ice and briefly washed with 70% ethanol before tissue dissection in 1X-PBS (Phosphate Buffered
Saline). Guts were fixed for 1 hour on a shaker at room temperature in fixative containing 4% paraformaldehyde (PFA) in Phosphate-
Buffered Saline (PBS). Samples were then washed 3 times for 20 minutes each in PBS, and incubated overnight in PBS/0.5% Triton
X-100 + 2% BSA, at 4 C. Staining with primary antibodies was carried out for 24 hours at 4 C in PBS/0.5% Triton X-100 + 2% BSA.
Samples were then washed 3 times for 20 minutes in PBS/0.5% Triton X-100, and incubated for 2 hours at room temperature with
secondary antibodies diluted in PBS/0.5% Triton X-100 + 2% BSA. Three 20-minute final washes were performed in PBS. Guts were
mounted between glass slides and coverslips (Electron Microscopy Sciences) using either Vectashield Antifade Mounting medium
with DAPI (Vector Laboratories) in the case of anti-FMR1 staining, or Prolong Gold Antifade mounting medium (Invitrogen) for other
stainings. All samples were imaged on a Leica SP8 confocal microscope (as described below).
Primary antibodies used were: rabbit anti-cleaved caspase 3 (1:100, Cell Signaling Technology), mouse anti-FMR1 (1:60, DSHB),
chicken anti-GFP (1:1000, Aves), mouse anti-gH2Av (1:40, DSHB), rabbit anti-dsRed that recognizes mCherry (1:100, Takara Bio).
Secondary antibodies used were: Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 488 donkey anti-rabbit, Alexa Fluor 555 donkey
anti-rabbit, Alexa Fluor 568 donkey anti-mouse, Alexa Fluor 568 donkey anti-rabbit (Invitrogen Molecular Probes, 1:1000); Alexa Fluor
488 donkey anti-chicken (Jackson ImmunoResearch Labs, 1:100).
Brain Immunohistochemistry
Adult males were anesthetized on ice and briefly washed with 70% ethanol before dissection in PBS. Dissected brains were fixed for
25 minutes on a shaker at room temperature, in fixative containing 4% PFA in PBS. Samples were then washed 3 times for 20 minutes
each in PBS/0.2% Triton X-100 and incubated overnight in PBS/0.2% Triton X-100 + 2% BSA at 4 C. Staining with primary antibodies
was carried out for 48 hours at 4 C in PBS/0.2% Triton X-100 + 2% BSA (72 hours when Bruchpilot staining was included).
Samples were then washed 3 times for 20 minutes each in PBS/0.2% Triton X-100, and incubated for 48 hours with secondary
antibodies diluted in PBS/0.2% Triton X-100 + 2% BSA. Finally, three 20-minute washes were performed using PBS/0.2% Triton
X-100 followed by a 20-minute wash in PBS. Brains were mounted between glass slides and coverslips (Electron Microscopy Sci-
ences) in Prolong Gold Antifade mounting medium (Invitrogen), and imaged on a Leica SP8 confocal microscope (as
described below).
Primary antibodies used were: chicken anti-GFP (1:1000, Aves), mouse anti-Bruchpilot (1:7, DSHB), rabbit anti-dsRed that recog-
nizes mCherry (1:100, Takara Bio). Secondary antibodies used were: Alexa Fluor 488 donkey anti-chicken (Jackson ImmunoRe-
search Labs, 1:100); Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 568 donkey anti-rabbit, Alexa Fluor 568 donkey anti-mouse,
Alexa Fluor 647 donkey anti-mouse, (Invitrogen Molecular Probes, 1:1000).
Expression Patterns of Gal4 and LexA Lines
Expression patterns of 11H05-Gal4, 60D04-Gal4, 50A07-LexA, and 68A07-LexA in the nervous system were visualized with a mem-
brane-targeted GFP, mCD8::GFP (Lee and Luo, 1999), stained with anti-GFP antibodies. Antibodies against Bruchpilot (Brp), a pre-
synaptic cytoskeletal protein (Wagh et al., 2006), were used to visualize the nervous system. The expression of these lines in the gut
was visualized with the bright nuclear fluorescent reporter RedStinger (Barolo et al., 2004) that did not require antibody staining.
Expression patterns of mex1-Gal4, myo1A-Gal4 and elav-Gal4 in the gut and the nervous system were visualized with nls-mCherry
(Caussinus et al., 2008) (stained with anti-dsRed antibodies that recognize mCherry). We used nls-mCherry here because expression
of RedStinger or nls-GFP in the whole nervous system seemed to be toxic to the flies, and we wanted the same marker in the gut and
the brain for direct comparison.
ROS imaging
In situ ROS detection was performed using either dihydroethidium (DHE) (Sigma-Aldrich) or 20 ,7’-dichlorofluorescein (H2DCF)
(Sigma-Aldrich) following previously described protocols (Owusu-Ansah et al., 2008; Rera et al., 2011). For DHE staining, flies
were anesthetized on ice and dissected in Schneider’s insect medium (GIBCO). Whole tissues were then incubated on a shaker
at room temperature with 60 mM DHE for 7 minutes in the dark. Before mounting, samples were washed three times for 5 minutes
in Schneider’s medium and once in PBS for 5 minutes, using a shaker at room temperature. For H2DCF, flies were anesthetized using
CO2 and dissected in PBS. Tissues were then incubated on a shaker at room temperature with 10 mM H2DCF for 15 minutes in the
dark. Before mounting, samples were washed three times for 5 minutes each in PBS using a shaker at room temperature.
Samples from each independent experiment were mounted between glass slides and coverslips (Electron Microscopy Sciences)
using Vectashield Antifade Mounting medium with DAPI (Vector Laboratories) and immediately imaged with identical settings on a
Leica SP8 confocal microscope (as described below).
LysoTracker staining
Lysosomal foci were detected using LysoTracker (Invitrogen) following a protocol from Ulgherait et al. (2014). Flies were anesthetized
on ice and dissected in cold PBS. Tissues were then incubated for 1.5 minutes, protected from light, in 1 mM LysoTracker Red at room
temperature on a shaker. Before mounting, five 30-s washes in PBS were performed at room temperature on a shaker. Samples were
mounted between glass slides and coverslips (Electron Microscopy Sciences) using Vectashield Antifade Mounting medium with
DAPI (Vector Laboratories), and imaged immediately with the same intensity settings on a Leica SP8 confocal microscope (as
described below).
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SYTOX Green necrosis assay

Necrosis was detected with SYTOX Green nucleic acid stain (Invitrogen) following a protocol from Lee et al. (2016). Flies were anes-
thetized on ice and dissected in cold PBS. Guts were incubated in 1 mM final concentration of SYTOX Green and 4% PFA for 40 mi-
nutes at room temperature on a shaker. Tissues were then washed three times for 20 minutes with PBS at room temperature on a
shaker, and mounted between glass slides and coverslips (Electron Microscopy Sciences) using Vectashield Antifade Mounting me-
dium with DAPI (Vector Laboratories). Samples were imaged on a Leica SP8 confocal microscope (as described below).
Fluorescent microscopy
Images were acquired with a Leica SP8 confocal microscope. A minimum of 7 samples per genotype was scored for each experiment
and at least 2 independent experiments were performed. Laser, filter and gain settings remained constant within each independent
experiment. Channels were scanned sequentially using these objectives: 10x for DHE (ROS) imaging and the expression pattern of
mex1-Gal4, elav-Gal4 and myo1A-Gal4 in the whole fly gut (nls-mCherry; Figures 7C and S7A); 20x oil for all other expression pat-
terns, gH2Av, cleaved caspase-3 and SYTOX Green imaging; 40x oil for FMR1 and LysoTracker imaging. Confocal Z stacks were
analyzed using Fiji, maximum projections are shown.
Image quantification
Image quantification was performed using Fiji. For quantification of total ROS levels, pixel intensities of Z stacks (sum slices) were
used. The mean of the summed DHE or H2DCF intensities averaged from each tissue was used for statistical analysis. For quanti-
fication of immunostainings, pixel intensities of Z stacks (sum slices) were used. The mean of the summed pixel intensities averaged
from each tissue and normalized to the area of selection was used for statistical analysis.
Mice
Sleep deprivation
For continuous sleep deprivation, mice were placed into a restriction chamber (Pinnacle Technology 9000-K-S) in which a rotating
bar, placed at short distance above the cage floor, is kept under constant but gentle motion to limit sleep. The bar was programed to
move at speed 7 and to alternate between clockwise and counter-clockwise rotations. Age-matched control non-deprived mice were
placed into a similar-sized chamber but without the bar rotating. Control and sleep-restricted mice were housed on autoclaved corn-
cob bedding (8B Bed-o’Cobs 1/8’’ 1.25 cu ft, Scott Pharma Inc% # 4B) where they had ad libitum access to food (Pico Lab Rodent
Diet 20 # 5053) and water. For each condition (control and sleep deprived), 5 males were placed in each cage 48 hours before the
beginning of the sleep deprivation protocol to allow for acclimation. For each independent experiment, one mouse was taken out
from each cage after 1, 2, and 5 days of sleep deprivation for dissections. At least 2 independent experiments were performed
for each mouse strain and for each tissue analyzed. Mouse weights were recorded over the deprivation period. Each day, the
same amount of food was provided to the sleep-restricted and non-restricted groups.
Sleep monitoring and scoring
To monitor sleep, a single male was placed in a restriction chamber (Pinnacle Technology 9000-K-S) equipped with a 3-channel teth-
ered EEG/EMG system (Pinnacle Technologies 8200-K1-iSL). Head-mounted preamplifiers (Pinnacle Technologies 8201) were im-
planted surgically. For electroencephalography/electromyography (EEG/EMG), mice were anesthetized with isoflurane and mounted
onto a stereotactic apparatus (Kopf) for surgery. The head was shaved, a 1.5 cm rostral-caudal incision was made starting from
approximately 3.5 mm anterior of bregma, and the EEG/EMG headmount was adhered onto the surface of the dry skull with Loctite
404 Instant Adhesive (Fisher Scientific #NC0619400). Screws (Pinnacle Technologies 8209, 8212) were drilled into the skull at the
corresponding holes in the headmount for hippocampal and cortical EEG recordings. To insert the EMG wires, small pockets in
the nuchal muscles were made. C&B Metabond Adhesive Luting Cement (Parkell) was applied to the headmount to protect and insu-
late EEG/EMG leads. The skin around the headmount was sutured and mice were allowed to recover for at least a week. Implanted
mice were tethered to the 3-channel EEG/EMG system in the restriction chamber and allowed to acclimate for 2 days before
recording. Recordings were acquired in standard conditions for 48 hours with no rotation of the bar, followed by 48 hours with
the bar rotating (and then by 24 hours with no rotation of the bar for rebound experiments).
Quantification of Sleep Restriction
Sleep recordings were scored in 10-s epochs over 48 hours of normal sleep and sleep restriction, with the sleep analysis software
Sirenia Sleep Pro. Analysis was semi-automated using both threshold scoring and cluster scoring. Non-REM EEG epochs were
scored as low frequency (0.5-4 Hz range), high amplitude (+/ 150-250 mV) delta waves with low-amplitude EMG waves. REM
EEG epochs contained mixed frequencies with predominantly theta waves (6-9 Hz range), low amplitude (+/ 50-100 mV) and flat
EMG waves. Wake epochs were scored as high frequency (8-50 Hz), low amplitude (+/ 50-100 mV) EEG waves with high-amplitude
EMG. Automated scored epochs were visually inspected to ensure that each epoch was scored according to our definitions of wake,
non-REM, and REM, and epochs that were not scored by the set rules were manually scored. Epochs that contained movement ar-
tifacts were removed.
Sleep rebound was measured after 2 consecutive days of sleep deprivation. Rebound sleep was calculated by subtracting the
amount of sleep (REM or NREM) on the day before deprivation from the amount of sleep on the day following deprivation.
ROS measurement
Control and sleep-deprived mice were sacrificed at 1, 2, or 5 days after the onset of sleep restriction. Tissues (small intestine, quad-
riceps muscle, heart, lungs, liver, kidneys, brain, large intestine, spleen, pancreas) were collected and placed in room temperature
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PBS before embedding in O.C.T (Tissue-Tek). Embedded tissues were immediately placed on dry ice and sectioned on a Leica cryo-
stat within 6 hours post-dissection. 30 mm sections were mounted on microscope glass slides (VWR) and incubated for 30 minutes at
37 C with 10 mM DHE (Sigma-Aldrich). Samples were washed three times for 5 minutes each with PBS before mounting. Sections
from each independent experiment were mounted between glass microscope slides and coverslips (VWR) with DAPI Fluoromount-G
(Southern Biotech), and imaged immediately with identical settings on a Leica SP8 confocal, or Olympus VS120 slide scanner (brain
sections only).
Immunohistochemistry
Control and sleep-deprived mice were sacrificed 2 or 5 days after the onset of sleep restriction. Mice were perfused with ice-cold
PBS followed by 4% PFA. The small and large intestines were dissected, post-fixed for 24 hours in 4% PFA followed by 20% sucrose
for cryoprotection, and embedded in O.C.T (Tissue-Tek). Embedded tissues were placed at 80 C until sectioning. 30 mm tissue
sections were obtained using a Leica cryostat and mounted on a microscope glass slide (VWR). Sections were permeabilized for
30 minutes in PBS/0.2% Triton X-100 and incubated for 2 hours in PBS/0.2% Triton X-100 + 5% normal goat serum (Jackson
ImmunoResearch Labs). Samples were then immunostained for 48 hours with primary antibodies at 4 C in PBS/0.2% Triton X-
100 + 5% normal goat serum. Three 20-minute washes were performed with PBS/0.2% Triton X-100 before incubation with second-
ary antibodies for 2 hours in PBS/0.2% Triton X-100 + 5% normal goat serum at room temperature. Three final 20-minute washes
were performed with PBS/0.2% Triton X-100, after which samples were mounted between glass microscope slides and coverslips
(VWR) with DAPI Fluoromount-G (Southern Biotech), and imaged on a Leica SP8 confocal microscope (as described below).
Primary antibodies used were: rabbit anti-cleaved caspase-3 (1:100, Cell Signaling Technology), mouse anti-gH2Ax (1:500, Cell
Signaling Technology), mouse anti-TIA1 (1:300, Santa Cruz). Secondary antibodies used were: Alexa Fluor 488 donkey anti-mouse,
Alexa Fluor 555 donkey anti-rabbit (Invitrogen Molecular Probes, 1:500).
Fluorescent microscopy
Samples were visualized and images acquired with either a Leica SP8 confocal microscope (all tissues except the brain for DHE, and
all immunostainings) or an Olympus VS120 slide scanner (brain sections only). A minimum of 12 samples per tissue was scored for
each independent experiment and at least 2 independent experiments were performed for each tissue. Laser, filter and gain settings
remained constant within each independent experiment, and channels were scanned sequentially using a 10x objective. Confocal Z
stacks and whole slide scanner images were analyzed using Fiji.
For quantification of total ROS levels, pixel intensities of Z stacks (sum slices) were used for all tissues except the brain (see below).
The mean of the summed DHE intensities from each sample was used for statistical analysis.
For ROS measurement in the brain, whole slide scanning was used and quantification was performed using pixel intensities of the
fluorescent signal. The mean DHE intensity from each sample was used for statistical analysis.
QUANTIFICATION AND STATISTICAL ANALYSIS
Statistical analyses were performed using Graphpad Prism software 7 (GraphPad Software Inc.), except for bootstrap analysis of
median survival (adjusted for multiple comparisons with Bonferroni’s method) that was done using a custom MATLAB software pro-
gram. Data are presented as mean and SEM unless indicated otherwise. Please see Table S1 for detailed statistical analysis of each
figure, including sample sizes and tests used.
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Supplemental Figures
Figure S1. A Second Fluorescent Reporter Also Indicates a ROS Increase in the Gut When Sleep Is Restricted; Expression of TrpA1 in the Gut
Does Not Cause ROS Accumulation, Related to Figures 1 and 2 Expression of TrpA1 in the gut does not cause ROS accumulation, Related to
Figures 1 and 2
(A) Oxidized H2DCF (H2DCF ox) reports an increase in ROS levels in the gut of sleep-deprived flies (blue) relative to the parental controls on day 10 of sleep
deprivation. Mean and SEM. (B) Expression of 11H05-Gal4 and 60D04-Gal4 in the gut, visualized with a nuclear fluorescent reporter RedStinger. The two Gal4s
label few cells in the anterior and posterior midgut (arrows). Guts outlined in white to help visualization. (C)-(H) Expression and activation of TrpA1 in different gut
cell populations (enterocytes (myo1A-Gal4, see also Figure S7) or intestinal stem cells and enteroblasts (esg-Gal4)) does not affect sleep (C) and (F), mean and
SEM (shading)); longevity (D) and (G), error bars omitted for clarity); or ROS levels (E) and (H), mean and SEM). Representative images. All scale bars, 200 mm. All
survival experiments were performed twice. Sample sizes and statistics, Table S1.
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Figure S2. Sleep Reduction and Shortened Lifespan in elav>CycARNAi Flies and ryeT227M, sssD40, and inc2 Mutants; Sleep Loss Is Not
Sustained in elav>incRNAi Animals; fmn Mutants Are Sleep-Deprived but Show No ROS Accumulation and Have Normal Lifespan, Related to
Figures 1, 2, and 3
(A)-(D) Reduced daily sleep and decreased lifespan in elav>CycARNAi flies (A), ryeT227M mutants (B), sssD40 mutants (C) and inc2 mutants (D). Sleep plots, mean
and SEM (shading); survival plots, error bars omitted for clarity. (E), (F) Sleep loss is not sustained in flies expressing incRNAi in neurons (elav>incRNAi). Mean and
SEM. (G), (H) Survival (error bars omitted for clarity) and ROS levels (mean and SEM) are normal in elav>incRNAi flies. (I), (J) fmn mutants experience severe sleep
loss (mean and SEM) but show no ROS increase (mean and SEM) in the gut and do not die prematurely (error bars omitted for clarity). Representative images are
shown. Scale bars, 200 mm. All survival experiments were performed 2-3 times. Sample sizes and statistics, Table S1.
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Figure S3. Oxidative Stress in the Gut Decreases When Sleep Deprivation Is Stopped; Gut Permeability Is Not Majorly Altered upon Sleep
Deprivation, Related to Figure 4
(A), (B) Levels of oxidative stress markers in the gut decrease after 15 days of recovery from sleep deprivation (SD). (C) The gut does not become leaky upon sleep
deprivation (i.e., absence of a ‘‘Smurf’’ phenotype). Aged UAS-TrpA1 fly used as positive control (the gut becomes leaky with age). (D) Left, daily food intake
increases in sleep-deprived flies (11H05>TrpA1 and 60D04>TrpA1) compared to the parental controls (11H05-Gal4, 60D04-Gal4, UAS-TrpA1). Right, blue dye
almost completely cleared from the gut of both sleep-deprived (11H05>TrpA1 and 60D04>TrpA1) and non-deprived (11H05-Gal4, 60D04-Gal4, UAS-TrpA1) flies
after 12 hours of feeding with regular (non-dyed) food. Representative images. Some guts are outlined in white to help visualization. Mean and SEM. Sample sizes
and statistics, Table S1.
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Figure S4. Sleep Deprivation Using a Second Mouse Strain Also Leads to ROS Accumulation in the Small and Large Intestines, Related to
Figure 5
(A) REM sleep, Non-REM sleep, and wakefulness in sleep-deprived (SD) and non-deprived (Non-SD) CBA/CaJ mice, as reported by EEG/EMG recordings. (B), (C)
DHE ox reports high levels of ROS in the small and large intestines of sleep-deprived CBA/CaJ mice. Brain: h, hippocampus; c, cortex. Representative images.
Scale bars, brain, 1 mm; other tissues, 200 mm. (D) When deprivation is stopped (here, after two days), sleep rebound (Non-REM and REM) is observed. (E) Body
weight (left) and food intake (right) of sleep deprived and non-deprived mice. Mean and SEM. Sample sizes and statistics, Table S1.
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Figure S5. Examples of Antioxidant Compound Treatments that Fail to Extend Survival and to Reduce ROS Levels in the Gut, Related to
Figure 6
(A) Example survival data for antioxidant treatments that failed to rescue survival of sleep-deprived flies (all such compounds were tested at several concen-
trations). Mean and SEM. (B) ROS levels remain high in the guts of sleep-deprived flies treated with the non-rescuing antioxidants. Representative images. Scale
bar, 200 mm. Mean and SEM. (C) No lifespan extension with antioxidants in non-deprived (UAS-TrpA1) flies. Left, population survival (error bars omitted for clarity);
right, survival data for individual flies (mean and SEM). All survival experiments were performed twice. Sample sizes and statistics, Table S1.
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Figure S6. Overexpression of Antioxidant Enzymes in the Gut Promotes Survival of Sleep-Deprived Animals, Related to Figure 7
(A) Expression of 68A07-LexA in the nervous system reported by mCD8::GFP. Brp, neuropil marker. (B) Sleep across three days. Day 1, 21 C: sleep amount is the
same between parental controls (68A07-LexA and LexAop-TrpA1) and animals expressing TrpA1 in neurons labeled by 68A07-LexA (68A07>TrpA1). When
temperature is raised to 29 C on day 2 (orange), 68A07>TrpA1 flies lost most of their sleep. In experiments involving LexAs, flies were kept in constant darkness
after temperature was raised to 29 C because sleep deprivation was more consistent than in light-dark cycles. Mean and SEM (shading). Right, daily sleep in
controls and experimental flies across lifespan. Mean and SEM. (C) Expression of 50A07-LexA and 68A07-LexA in the gut, visualized with a nuclear fluorescent
reporter RedStinger. The two LexA lines label few cells in the anterior and posterior midgut (arrows). Guts outlined in white to help visualization. (D) Survival of
sleep-deprived (red and purple) and non-deprived animals at 29 C in constant darkness. Error bars omitted for clarity. (E) Daily food intake is not different in sleep-
deprived flies (50A07>TrpA1) compared to parental controls (LexAop-TrpA1 and 50A07-LexA). Mean and SEM. (F) Overexpressing antioxidant enzymes (SOD1
or SOD2) in neurons throughout the nervous system using elav-Gal4 fails to prevent ROS accumulation in the gut. Mean and SEM. (G) Extended survival of sleep-
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deprived flies (68A07>TrpA1) expressing transgenic antioxidant enzymes (SOD1, SOD2 or CAT) in the gut with myo1A-Gal4. Left, survival for individual flies
(mean and SEM); right, population survival (error bars omitted for clarity). (H) Daily sleep across lifespan (left, error bars omitted for clarity) or during the first
18 days of sleep deprivation (the time it took for the sleep-deprived flies to die if they had no transgenic expression of antioxidant enzymes) (right, mean and SEM)
of sleep-deprived flies (68A07>TrpA1) expressing transgenic antioxidant enzymes (SOD1, SOD2 or CAT) in the gut. Representative images. Scale bars, 200 mm.
All survival experiments were performed 2-3 times. Sample sizes and statistics, Table S1.
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Figure S7. Overexpression of Antioxidant Enzymes in the Gut Prevents Oxidative Damage in This Tissue and Promotes Survival upon Sleep
Deprivation, Related to Figure 7
(A) Expression of myo1A-Gal4 in the gut and the central nervous system, visualized with nls-mCherry. (B) Survival of sleep-deprived flies expressing transgenic
antioxidant enzymes (SOD1, SOD2 or CAT) in the gut with myo1A-Gal4. Dot plot, survival for individual flies (mean and SEM); curves, population survival (error
bars omitted for clarity). Survival experiments were performed at least 3 times. (C) Daily sleep of sleep-deprived flies expressing transgenic antioxidant enzymes
(SOD1, SOD2 or CAT) in the gut with myo1A-Gal4 (left) or mex1-Gal4 (right). Sleep is shown either across the entire lifespan (top, error bars omitted for clarity) or
during the first 11 days of sleep deprivation (the time it took for the sleep-deprived flies to die if they had no transgenic expression of antioxidant enzymes)
(bottom, mean and SEM). (D) ROS accumulation is prevented by transgenic expression of antioxidant enzymes (SOD1, SOD2 or CAT) in the gut with mex1-Gal4.
(E) Overexpressing the antioxidant enzyme SOD1 in the gut with mex1-Gal4 (50A07>TrpA1, mex1>SOD1) prevents DNA damage and cell death in this tissue. (F),
(G) ROS accumulation is prevented by transgenic expression of antioxidant enzymes (SOD1, SOD2 or CAT) in the gut with myo1A-Gal4. (F), (H) Overexpression of
SOD1 in the gut of sleep-deprived flies using myo1A-Gal4 (50A07>TrpA1, myo1A>SOD1) prevents DNA damage and cell death in this tissue. Representative
images. Scale bars, 200 mm. Guts outlined in white to help visualization. Mean and SEM. Sample sizes and statistics, Table S1.

Sleep Loss Can Cause Death Through Accumulation of Reactive Oxygen Species in The Gut

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Sleep Loss Can Cause Death Through Accumulation of Reactive Oxygen Species in The Gut

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Sleep Loss Can Cause Death through Accumulation

d Gut-accumulated ROS trigger oxidative stress in this organ

d Preventing ROS accumulation in the gut allows survival

Vaccaro et al., 2020, Cell 181, 1–22

Cell 181, 1–22, June 11, 2020 ª 2020 Elsevier Inc. 1

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2 Cell 181, 1–22, June 11, 2020

oxidation after long-term sleep restriction (Everson et al., 2014; RESULTS

Figure 1. Sleep Deprivation Shortens Lifespan

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6 Cell 181, 1–22, June 11, 2020

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8 Cell 181, 1–22, June 11, 2020

Figure 4. Sleep Deprivation Causes Oxidative Stress in the Gut

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10 Cell 181, 1–22, June 11, 2020

Figure 5. ROS Accumulate in the Mouse Gut upon Sleep Deprivation

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12 Cell 181, 1–22, June 11, 2020

Figure 6. Clearance of ROS Prevents Death of Thermogenetically Sleep-Deprived Flies

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16 Cell 181, 1–22, June 11, 2020

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18 Cell 181, 1–22, June 11, 2020

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20 Cell 181, 1–22, June 11, 2020

Cell 181, 1–22, June 11, 2020 21

22 Cell 181, 1–22, June 11, 2020

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Cell 181, 1–22.e1–e7, June 11, 2020 e1

e2 Cell 181, 1–22.e1–e7, June 11, 2020

Data and Code Availability

EXPERIMENTAL MODELS AND SUBJECT DETAILS

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e4 Cell 181, 1–22.e1–e7, June 11, 2020

Cell 181, 1–22.e1–e7, June 11, 2020 e5

SYTOX Green necrosis assay

e6 Cell 181, 1–22.e1–e7, June 11, 2020

QUANTIFICATION AND STATISTICAL ANALYSIS

Cell 181, 1–22.e1–e7, June 11, 2020 e7

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